Composition comprising the polyprotein ns3/ns4 and the polypeptide ns5b of hcv, expression vectors including the corresponding nucleic sequences and their therapeutic use

ABSTRACT

The invention relates to a peptidic compound containing a polyprotein NS3/NS4 of a hepatitis C virus and a polypeptide NS5 b  of hepatitis C virus. Said invention also relates to expression vectors such as adenovirus and poxvirus in which nucleic sequences coding for the polyprotein NS3/NS4 and the polypeptide NS5 b . The inventive compound can be used for a therapeutic application.

The present invention relates to the field of prophylactic andtherapeutic vaccination directed against the hepatitis C virus (HCV). Itrelates in particular to a novel composition containing a polyproteincorresponding to the two collinear proteins NS3 and NS4 (hereaftercalled polyprotein NS3/NS4) and a polypeptide constituted by NS5b, thevectors, such as adenovirus or poxvirus, capable of expressing thiscomposition and their use as vaccine.

Hepatitis C is the major cause of transfusion-acquired hepatitis.Hepatitis C can also be transmitted by other percutaneous routes, forexample by injection of drugs by intravenous route. The risk ofcontamination of health professionals is moreover not negligible. Sexualtransmission has been described.

Hepatitis C differs from other forms of liver diseases associated withviruses, such as hepatitis A, B or D. The infections by the hepatitis Cvirus (HCV or HCV) are mostly chronic resulting in diseases of theliver, such as hepatitis, cirrhosis and carcinoma in a large number ofcases (5 to 20%) and represents 30% of the hepatic transplants indeveloped countries.

Although the risk of transmission of the virus by transfusion hasdiminished owing to the introduction of screening tests in the 1990s,the frequency of new HCV infections remains high. By way of example, arecent study indicates that today there are still 10,000 to 15,000 newcases of infection per year in France (S. Deuffic et al., Hepatology1999; 29: 1596-1601). Currently, approximately 170 million peopleworldwide are chronically infected by HCV (Hepatitis C: Globalprevalence (update), 2000, Weekly Epidemiological Record, Vol 75(3)).The high-risk populations are principally hospital staff andintravenous-drug users, but there are asymptomatic blood donors who donot belong to these high-risk groups and in whom circulating anti-HCVantibodies have been found. For the latter, the infection route has notyet been identified. HCV infections therefore exist (estimated atbetween 5 and 10%), known as sporadic infections, the etiology of whichis unknown and which cannot be controlled.

HCV was the first hepatotropic virus isolated by means of molecularbiology techniques. The viral genome sequences were cloned before theviral particle was visualized.

HCV belongs to a new genus of the Flaviviridae family, thehepaciviruses. It is a positive single-strand RNA virus, of 9.5 kb,which is replicated by a complementary RNA copy and the translationproduct of which is a polyprotein precursor of approximately 3,000 aminoacids. The 5′ end of the HCV genome corresponds to an untranslatedregion adjacent to the genes that code for the structural proteins, thecore protein of the nucleocapsid, the two envelope glycoproteins, E1 andE2, and a small protein called p7. The 5′ untranslated region and thegene core are relatively well preserved in the different genotypes. Theenvelope proteins E1 and E2 are encoded by regions that are morevariable from one isolate to another. The protein p7 is an extremelyhydrophobic protein, which may constitute an ion channel. The 3′ end ofthe HCV genome contains the genes that code for the non-structuralproteins (NS2, NS3, NS4, NS5) and for a 3′ non-coding region possessinga well-conserved domain (Major M E, Feinstone S M, Hepatology, June1997, 25 (6): 1527-1538).

At present, the most effective therapy for the treatment of hepatitis Ccombines pegylated interferon and ribavin (Manns M P et al., The Lancet,22 Sep. 2001, Vol. 358, 958-965). Whilst this therapy is particularlyeffective in the case of patients infected by viral strains belonging tothe genotypes 2 and 3, it still has only a limited effect on thegenotypes 1a, 1b and 4 (Manns M P, op. cit.). Less than 50% of thetreated patients become “long-term responders”. Moreover, this therapyis an expensive intervention (10,000 to 15,000 euros/patient/year) andis associated with toxic effects. In fact, 5 to 10% of the patients areobliged to stop treatment before the end.

It is therefore necessary to develop a vaccine composition targeting allthe genotypes.

Several studies now show that the control of an infection caused by HCVeither naturally (spontaneous resolution), or after treatment(therapeutic resolution) is associated with the induction orpotentialization of cell-mediated immune responses involving the T-CD4⁺and T-CD8⁺ lymphocytes (as described for example in LECHNER, F. et al.,Eur. J. Immunol., 30: 2479-2487 (2000) and in Thimme R. et al., 2001, J.Exp. Med., 194 (10): 1395-1406).

The molecules of the major histocompatibility complex (MHC, also knownas HLA in humans) are referred to as class I or class II. The class Imolecules are expressed on virtually all of the nucleated cells and areable to present epitopes or peptides to the CD8⁺ cytotoxic T lymphocytes(CTL). The class II molecules are able to present epitopes to the CD4⁺ Tcells, but their expression is restricted to antigen-presenting cells.

The vaccines against the hepatitis C virus currently envisaged are basedon the use of adjuvant recombinant proteins, peptides, expressionvectors among which there can be mentioned vectors of viral or bacterialorigin or of naked DNA. In this case, one or more viral proteins or oneor more genes coding for these viral proteins are used.

When several viral proteins or one or more genes coding for these viralproteins are selected, the latter are often constituted either by someor all of the structural proteins (Makimura et al., 1996, Vaccine, 14:28-34; Fournilier A. et al., 1999, J. Virology, 73: 7497-7504), or byindividual non-structural proteins or comprising at least two contiguousproteins (Brinster et al., 2001, Hepatology, 34: 1206-1217), or by amixture of structural and non-structural proteins (Pancholi et al.,2003, J. Virology, 77: 382-390).

The Patent Application WO99/38880 describes the use of three genescoding separately for the three proteins NS3, NS4 and NS5 (a and b) in avaccine composition comprising three DNA vaccines each expressing thesethree proteins separately. The authors show the induction of Tlymphocytes specific to the three antigens in mice. Only the vaccineexpressing NS5a and b has been tested in vivo in a protection test.

The Patent Application WO01/30812 describes the use of a fusion proteinconstituted by the non-structural proteins NS3, NS4 and NS5a, ifnecessary in combination with the non-structural protein NS5b. Theauthors have indicated that this combination made it possible toactivate the HCV-specific T cells. This patent application simplydescribes the ability of vaccine formulations (naked-DNA,recombinant-adenovirus or recombinant-vaccinia-virus type) expressingthe fusion protein NS3, NS4, NS5a or the protein NS5a to induce specificimmune responses mediated by specific T lymphocytes.

The Applicant has now demonstrated, against all expectation, that theparticular combination of the non-structural proteins NS3, NS4 and NS5b,NS3 and NS4 being expressed colinearly had a better immunogenic powerand protective power superior to that obtained with a vaccine alsoincluding, apart from these non-structural proteins, the protein NS5aand/or other structural proteins of HCV such as core, E1 or E2, and hadan effect on the ability of cells originating from patients infected byviral strains to induce specific immune responses.

Thus, an object of the present invention is a peptide compositioncomprising a polyprotein NS3/NS4 of the hepatitis C virus, as well as apolypeptide NS5b of the hepatitis C virus.

An object of the invention is also the vectors including the nucleotidesequences coding for this peptide composition, such as the adenovirusesand poxviruses, as well as microorganisms or host cells transformed bythese vectors.

An object of the invention is finally the antibodies directed againstthe peptide composition of the invention, as well as the use of thepeptide composition, vectors and antibodies for the preparation of amedicament intended for the inhibition or control of an infection causedby the hepatitis C virus, and in a vaccine composition.

The present invention therefore proposes a novel peptide compositionconstituted by a polyprotein NS3/NS4 and a polypeptide NS5b of HCV,which composition has the ability to stimulate a cell-mediated immuneresponse specific to HCV, such that it is useful in the field ofprophylactic and therapeutic vaccination directed against the hepatitisC virus.

The polyprotein NS3/NS4 of the peptide composition of the invention isconstituted by the protein NS3 and the protein NS4a and b, withoutinterruption in the peptide sequence, as in the native polyprotein. Infact, as indicated previously, the HCV genome contains a single openreading frame that is transcribed into a polyprotein. This HCVpolyprotein can be cleaved in order to produce at least ten distinctparts, in the order NH₂-Core-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH.

The protein NS3 is a protein of 630 amino acids, which appearsapproximately from amino acid 1027 to amino acid 1657 of thepolyprotein. The protein NS4, a protein of 314 amino acids, appearsapproximately from amino acid 1658 to amino acid 1972 (numbering withrespect to HCV-1) (Choo et al., 1991, Proc. Natl. Acad. Sci., vol 88:2451-2455). The polyprotein NS3/NS4 therefore appears approximately fromamino acid 1027 to amino acid 1972.

As regards the polypeptide NS5b also contained in the composition of theinvention, it is constituted by 590 amino acids and appearsapproximately from amino acid 2421 to amino acid 3011 of the polyprotein(Choo et al., 1991, op. cit.).

The protein NS3 comprises two distinct structural domains, namely anN-terminal domain endowed with an active serine protease activity thatis involved in the maturation of the viral polyprotein, and a C-terminaldomain comprising a helicase activity associated with an NTPase activitythat plays a role in the replication of the viral genome.

By “polyprotein NS3/NS4” and “polypeptide NS5b”, is of course meant thepolyproteins and polypeptides having the native amino acid sequences,originating from any HCV strain and isolate, as well as their analogues,muteins and homologues.

By “analogues” or “muteins” of the polyprotein and of the polypeptide,is meant the biologically active derivatives of the reference moleculesthat have the desired activity, namely the ability to stimulate acell-mediated immune response as defined above.

Generally, the term “analogue” refers to compounds having a nativepolypeptide sequence and structure having one or more additions,substitutions (generally conservative in terms of nature) and/or aminoacid deletions, relative to the native molecule, to the extent that themodifications do not destroy the immunogenic activity. By the term“mutein”, is meant the peptides having one or more elements imitatingthe peptide (peptoids), such as those described in the PatentApplication PCT WO91/04282. Preferably, the analogue or the mutein haveat least the same immunoactivity as the native molecule. Processes forpreparing polypeptide analogues and muteins are known to a personskilled in the art and are described below.

The particularly preferred analogues include substitutions that areconservative in nature, i.e. the substitutions, which take place in afamily of amino acids. Specifically, the amino acids are generallydivided into 4 families, namely (1) the acid amino acids such asaspartate and glutamate, (2) the basic amino acids such as lysine,arginine and histidine, (3) the non-polar amino acids such as alanine,leucine, isoleucine, proline, phenylalanine, methionine and tryptophaneand (4) the polar non-charged amino acids such as glycine, asparagine,glutamine, cysteine, serine, threonine and tyrosine. Phenylalanine,tryptophane and tyrosine are sometimes classified as aromatic aminoacids. For example, it can reasonably be predicted that an isolatedreplacement of leucine by isoleucine or valine, of an aspartate by aglutamate, of a threonine by a serine, or a similar conservativereplacement of one amino acid by another amino acid having a structuralrelationship, will not have a major effect on the biological activity. Aperson skilled in the art will easily determine the regions of thepeptide molecule of interest that can tolerate a change by referring tothe Hopp/Woods and Kyte-Doolittle plots, well known in the art.

By “homology”, is meant the percentage of identity between two peptidemolecules, such as polyproteins and polypeptides. Two amino acidsequences are “more or less homologous” to each other when the sequenceshave at least 60%, preferably at least 75%, more preferably also atleast 80-85%, more preferably also at least 90% and still morepreferably at least 95-98% or more of sequence identity over a definedlength of the peptide molecules.

Generally, the term “identity” refers to an exact amino acid to aminoacid correspondence of two peptide sequences. The percentage of identitycan be determined by a direct comparison of the sequence informationbetween two molecules by aligning the sequences, counting the exactnumber of mismatches between the two aligned sequences, dividing by thelength of the shorter sequence and multiplying the result by 100. Thepercentage of identity can also be determined using computer programssuch as ALIGN, Dayhoff, M. O. in Atlas of Protein Sequence and StructureM. O. Dayhoffed., 1981, 5 Suppl., 3: 482-489.

The nucleic acid and amino acid sequences of a certain number of HCVstrains and isolates, and in particular of the protein NS3, of theprotein NS4 and of the polypeptide NS5b, have already been determined.

For example, the isolate HCV-J1 is described in Okamoto H. et al., 1992,Nucleic Acids Res., 20: 6410-6410. The complete coding sequences of twoindependent HCV isolates, namely the isolates HCV-J and -BK, have beendescribed in Kato et al., 1990, Proc. Natl. Acad., Sci., 87: 9524-9528and in Takamizawa et al., 1991, J. Virol., 65: 1105-1113 respectively.As regards the isolate HCV-1, it is described in Choo et al., 1990,Brit. Med. Bull., 46: 423-441 and in Choo et al., 1991, op. cit. Theisolate HVC-H has been described in Inchauspe G. et al, 1991, Proc.Natl. Acad. Sci., 88: 10292-10296. The isolate HCV-G9 has been describedin Okamoto H., et al., 1994, J. Gen. Virol., 45: 629-635. The isolatesHCV-J6 and -J8 have been described in Okamoto H., et al., 1991, J. Gen.Virol., 72: 2697-2704 and Okamoto H., et al., 1992, Virology, 188:331-341 respectively. The isolate HVC-BEBE1 has been described in NakoH., et al., 1996, J. Gen. Virol., 141: 701-704 and the isolate HCV-NZL1has been described in Sakamoto M., et al., 1994, J. Gen. Virol., 75:1761-1768. As regards the isolate HCV-Tr, it has been described inChayama K., et al., 1994, J. Gen. Virol., 75: 3623-3628. The isolatesHCV-ED43 and -EUH1480 have been described in Chamberlain R. W., et al.,1997, J. Gen. Virol., 78: 1341-1347 and Chamberlain R. W., et al., 1997,Biochem. Biophys. Res. Commun., 236: 44-49 respectively. The isolateHCV-EUHK2 has been described in Adams A., et al., 1997, Biochem.Biophys. Res. Commun., 234: 393-396. The isolates HCV-VN235, -VN405 and-VN004 have been described in Tokita H., et al., 1998, J. Gen. Virol.,79: 1847. Finally, as regards the isolates HCV-JK049 and -JK046, theyhave been described in Tokita H. et al., 1996, J. Gen. Virol., 77:293-301.

The HCV strains and isolates, as illustrated above, can have differentgenotypes, namely genotypes 1a (isolates HCV-1, -J1 and -H), 1b(isolates HCV-J and BK), 1c (isolate HCV-G9), 2a (isolate HCV-J6), 2b(isolate HCV-J8), 2c (isolate HCV-BEBE1), 3a (isolate HCV-NZL1), 3b(isolate HCV-Tr), 4a (isolate HCV-ED43), 5a (isolate HCV-EUH1480), 6a(isolate HCV-EUHK2), 7b (isolate HCV-VN235), 8b (isolate HCV-VN405), 9a(isolate HCV-VN004), 10a (isolate HCV-JK049) and 11a (isolateHCV-JK046).

According to one embodiment of the invention, NS3 and/or NS4 and/or NS5boriginate from viruses of different genotypes.

According to another embodiment, NS3 and/or NS4 and/or NS5b originatefrom viruses of the same genotype, preferably of genotype 1b.

The polyprotein NS3/NS4 and the polypeptide NS5b contained in thepeptide composition of the invention can be either of native origin, orof recombinant origin.

The polyprotein NS3/NS4 and the polypeptide NS5b of native origin areobtained from HCV strains or isolates, by means of the use of syntheticoligonucleotide primers that will serve to amplify the native viralsequences, either from sera of patients infected by the targeted viralgenotype or genotypes, or from already purified viral RNA, originatingfor example from patients' blood or liver, or from complementary DNAthat is free or cloned beforehand in an expression vector, or also fromviral particles purified from biological samples or in vitro propagationsystem.

The polyprotein NS3/NS4 and the polypeptide NS5b of the invention ofrecombinant origin can also be obtained by the genetic engineeringtechnique, which comprises the steps of:

culture of a microorganism or of eukaryotic cell(s) transformed using anucleotide sequence coding for said polyprotein NS3/NS4 or for saidpolypeptide NS5b and

recovery of the peptide produced by said microorganism or saideukaryotic cells.

This technique is well known to a person skilled in the art. For moredetails concerning this, reference can be made to the following work:Recombinant DNA Technology I, Editors Ales Prokop, Raskesh K Bajpai;Annals of the New-York Academy of Sciences, Volume 646, 1991.

The nucleotide sequences coding for the polyprotein NS3/NS4 and thepolypeptide NS5b can be prepared by chemical synthesis in conjunctionwith a genetic engineering approach or by genetic engineering alone,using the techniques well known to a person skilled in the art anddescribed for example in Sambrook J. et al., Molecular Cloning: ALaboratory Manual, 1989.

The nucleotide sequences coding for the polyprotein NS3/NS4 and thepolypeptide NS5b can be inserted into expression vectors in a suitableexpression system, in order to obtain the peptide composition of theinvention.

Of course, the nucleotide sequences can be inserted into a singleexpression vector or into two different expression vectors. In thelatter case, the sequence coding for the polyprotein NS3/NS4 is insertedinto one of the two vectors and the sequence coding for the polypeptideNS5b is inserted into the other vector, these two vectors being eitheridentical or different in nature.

Thus, another object of the invention is the expression vectorscomprising a nucleotide sequence coding for the polyprotein NS3/NS4 anda nucleotide sequence coding for the polypeptide NS5b, as well as themeans necessary to its expression.

By means necessary to the expression of a peptide is meant, the termpeptide being used for any peptide molecule, such as protein,polyprotein, polypeptide, etc., any means that make it possible toobtain the peptide, such as in particular a promoter, a transcriptionterminator, a replication origin and preferably a selection marker.

The means necessary to the expression of a peptide are operationallylinked to the nucleic acid sequence coding for the peptide of interest.By “operationally linked”, is meant a juxtaposition of said elementsnecessary to the expression and of the gene coding for the peptide ofinterest, which are in a relationship such that it is possible for themto function in an expected manner. For example, additional bases canexist between the promoter and the gene of interest to the extent thattheir functional relationship is preserved.

The means necessary to the expression of a peptide can be homologousmeans, i.e. included in the genome of the vector used, or beheterologous. In the latter case, said means are cloned with the peptideof interest to be expressed.

Examples of heterologous promoters include (i) the viral promoters suchas the SV40 promoter (simian virus 40), the promoter of thethymidine-kinase gene of the herpes simplex virus (TK-HSV-1), the LTR ofthe Rous sarcoma virus (RSV), the immediate first promoter of thecytomegalovirus (CMV) and the adenovirus major last promoter (MLP), aswell as (ii) any cell promoter that controls the transcription of thegenes coding for peptides in upper eukaryotes, such as the constitutivepromoter of the diphosphoglycerate-kinase gene (PGK) (Adra et al., 1987,Gene, 60: 65-74), the promoter of the liver-specific alpha-1 antitrypsinand FIX genes and the SM22 promoter specific to the smooth muscle cells(Moessler et al., 1996, Development, 122: 2415-2425).

According to one embodiment of the invention, the nucleotide sequencescoding for said polyprotein NS3/NS4 and said polypeptide NS5b originatefrom different genotypes.

According to another embodiment, the nucleotide sequences coding forsaid polyprotein and said polypeptide originate from a virus of the samegenotype, preferably genotype 1b.

Here too, by “nucleotide sequence” is meant all the sequences coding forthe native polyprotein NS3/NS4 and the native polypeptide NS5b, as wellas for their analogues, muteins and homologues, as defined previously.

Said sequences contained in the expression vector can be directlyinterlinked under the control of a single promoter and/or of a singleexpression-regulating element, or they can be separate, each beingdependent on expression promoters and/or regulators that are independentidentical or different.

As expression vectors that are suitable for the purposes of theinvention, there can be mentioned for example plasmids, adenovirus-typeviral vectors, poxviruses, vaccinia viruses, baculoviruses,salmonella-type bacterial vectors, BCG.

Adenoviruses have been detected in numerous animal species, do notintegrate and are only slightly pathogenic. They are capable ofinfecting a variety of cell types, cells in division and cells at rest.They possess a natural tropism for the bronchial epithelia. Moreover,they have been used as live enteric vaccines for many years with anexcellent safety profile. Finally, they can easily be made to grow andbe purified in large amounts. These characteristics have meant that theadenoviruses are particularly appropriate for use as expression vectorsand in particular as gene therapy vectors for therapeutic purposes andfor vaccines.

According to a preferred embodiment, the vector of the invention is anadenovirus.

Examples of adenoviruses to be used in the present invention can bederived from any source of human or animal origin, in particular ofcanine origin (for example CAV-1 or CAV-2; reference Genbank CAV1GENOMand CAV77082 respectively), of avian origin (reference GenbankAAVEDSDNA), of bovine origin (such as BAV3, Seshidhar Reddy et al.,1998, J. Virol., 72: 1394-1402), of ovine, feline, porcine origin, ofsimian origin, or from one of their hybrids. Any serotype can be used.However, adenoviruses of human origin are preferred and in particularadenovirus 5 (AdIV).

Generally, the mentioned viruses are available from the ATCC collectionsand have been the subject of numerous publications describing theirsequence, their organization and their biology, which allows a personskilled in the art to use them easily. For example, the sequence of theadenovirus type 5 is described in the Genbank database (M73260 andM29978) and is incorporated here by way of reference.

The genome of the adenovirus is constituted by a double-strand linearDNA molecule of approximately 36 kb carrying more than approximately 30genes necessary for terminating the viral cycle. The first genes aredivided into 4 regions dispersed in the genome of the adenovirus (E1 toE4). The E1, E2 and E4 regions are essential for viral replication. TheE3 region is considered as a non-essential region on the basis of theobservation that mutant viruses appear naturally or the hybrid viruseshaving lost this E3 region continue to replicate like wild-type virusesin cultured cells (Kelly and Lewis, 1973, J. Virol., 12: 643-652). Thelast genes (L1 to L5) mostly code for the structural proteinsconstituting the viral capsid. They overlap at least in part the firsttranscription units and are transcribed from a single promoter (MLP forMajor Late Promoter). Moreover, the adenoviral genome carries at the twoends of the cis-acting regions essential for DNA replication, the 5′ and3′ inverted terminal repeats (ITRs) and a packing sequence respectively.

The adenoviruses currently used in gene therapy protocols are strippedof the majority of the E1 region, which renders the viruses deficient atthe level of their replication in order to avoid their dissemination inthe environment and in the host organism. Moreover, most of theadenoviruses are also stripped of the E3 region in order to increasetheir cloning capacity. The feasibility of gene transfer using thesevectors has been demonstrated in a variety of tissues in vivo (see forexample Yei et al., 1994, Hum. Gene Ther., 5: 731-744; Dai et al., 1995,Proc. Natl. Acad Sci. USA, 92: 1401-1405; U.S. Pat. No. 6,099,831; andU.S. Pat. No. 6,013,638).

Preferably, the promoters used in the adenoviruses as expression vectorsare heterologous promoters such as the CMV and SV40 promoters.

Preferably also, the CMV promoter is the promoter of the polyproteinNS3/NS4 and the expression vector comprises as nucleotide sequencecoding for said polyprotein the expression cassette CMV-NS3-NS4.

By “expression cassette”, is meant a DNA sequence containing a promoterand an open reading frame for the expression of the peptide of interest,to be inserted into a vector.

Preferably also, the SV40 promoter is the promoter of the polypeptideNS5b and the expression vector comprises as nucleotide sequence codingfor said polypeptide the expression cassette SV40-NS5b.

According to one embodiment of the invention, the genome of theadenovirus is modified so as to replace the E1 region by the expressioncassette CMV-NS3-NS4 and to replace the E3 region by the expressioncassette SV40-NS5b.

The methods of suppression and of insertion of DNA sequences intoexpression vectors are widely known to a person skilled in the art andconsist in particular of steps of enzymatic digestion and ligation.

Another expression vector particularly appropriate for the purposes ofthe invention is a poxvirus, which constitutes another embodiment of theinvention.

The poxviruses constitute a group of enveloped complex viruses,differing principally in their unusual morphology, their large DNAgenome and their cytoplasmic replication site. The genome of severalelements of the poxviridae, comprising the Copenhagen strain of thevaccinia virus (VV) (Goebel et al., 1990, Virol. 179: 247-266 and517-563) and the modified vaccinia virus Ankara (MVA) strain (Antoine etal., 1998, Virol., 244: 635-396), has been mapped and sequenced. The VVstrain possesses a double-strand DNA genome of approximately 192 kbcoding for approximately 200 proteins approximately 100 of which areinvolved in the assembly of the virus. The MVA strain is a highlyattenuated strain of vaccinia virus, generated by more than 500 passagesin series of the vaccinia virus Ankara strain (CVA) over chicken embryofibroblasts (Mayr et al., 1975, Infection, 3: 6-16). The MVA virus hasbeen deposited in the Collection Nationale de Cultures deMicroorganismes (CNCM) under Number I-721. The determination of thecomplete sequence of the MVA genome and comparison with that of the VVallows precise identification of the alterations that have appeared inthe viral genome and the definition of seven deletions (I to VII) and ofnumerous mutations leading to fragmented open reading frames (Antoine etal., 1998, Virology, 244: 365-396).

Other examples of poxviruses that are appropriate for the purposes ofthe invention include duck pox, fowl pox, cow pox, entomopox, monkeypox, swine pox and penguin pox.

The poxvirus is found in two morphologically distinct forms, calledintracellular mature virus (IMV) and enveloped extracellular virus(EEV).

The poxvirus used as an expression vector of the invention has at leastone of the following characteristics, taken alone or in combination:

-   -   (i) the poxvirus is an MVA virus,    -   (ii) the poxvirus is in the IMV morphological form, and    -   (iii) the genome of the poxvirus is modified so as to insert the        expression cassette NS3/NS4 and to insert the expression        cassette NS5b.

When the genome of the poxvirus is modified so as to insert the twocassettes of interest, the means necessary to their expression arehomologues. Thus, in the case where the MVA virus is used, theexpression of NS3/NS4 can be for example under the control of thepromoter ph5r so that the corresponding expression cassette isph5r-NS3-NS4, and the expression of NS5b can be for example under thecontrol of the promoter p7.5 so that the corresponding expressioncassette is p7.5-NS5b, and vice versa.

According to a particular embodiment, when the genome of the poxvirus ismodified so as to insert the two cassettes of interest, the two saidexpression cassettes are oriented in the same direction.

According to another particular embodiment, they are oriented in theopposite direction.

Here too, the expression cassettes are inserted into the genome of thepoxvirus in a manner known to a person skilled in the art, as indicatedpreviously.

The vectors of the invention can also comprise sequences necessary fortargeting peptides towards particular cell compartments. An example oftargeting can be the targeting towards the endoplasmic reticulumobtained using address sequences of the leader sequence type originatingfrom the protein E3 of the adenovirus (Ciernik I. F., et al., TheJournal of Immunology, 1999, 162, 3915-3925).

They can also comprise sequences necessary for targeting towards thedendritic cells and for targeting at the membrane of the cells.

An object of the invention is also the microorganisms and the eukaryoticcells transformed by an expression vector of the invention.

By way of examples of microorganism that are suitable for the purposesof the invention, there can be mentioned the yeasts, such as those ofthe following families: Saccharomyces, Schizosaccharomyces,Kluveromyces, Pichia, Hanseluna, Yarrowia, Schwantomyces,Zygosaccharomyces, Saccharomyces cerevisiae, Saccharomycescarlsbergensis and Kluveromyces lactis being preferred; and thebacteria, such as E. coli and those of the following families:Lactobacillus, Lactococcus, Salmonella, Streptococcus, Bacillus andStreptomyces.

By way of examples of eukaryotic cells, there can be mentioned cellsoriginating from animals such as mammals, reptiles, insects andequivalent. The preferred eukaryotic cells are cells originating fromthe Chinese hamster (CHO cells), monkey (COS and Vero cells), babyhamster kidney (BHK cells), pig kidney (PK 15 cells) and rabbit kidney(RK13 cells), human osteosarcoma cell lines (143 B), HeLa human celllines and the human hepatoma cell lines (Hep G2-type cells), as well asinsect cell lines (for example of Spodoptera frugiperda).

The host cells can be provided in cultures in suspension or in flasks,in tissue cultures, organ cultures and equivalent. The host cells canalso be transgenic animals.

The invention also relates to antibodies directed against one of thepeptide compositions of the invention as defined previously or againstone of the expression vectors of the invention as defined previously.

The antibodies according to the invention are either polyclonal ormonoclonal antibodies.

The abovementioned polyclonal antibodies can be obtained by immunizationof an animal with the peptide composition of the invention or with thevector of the invention as “antigen of interest”, followed by therecovery of the antibodies sought in purified form, by sampling theserum of said animal, and separation of said antibodies from the otherconstituents of the serum, in particular by affinity chromatography on acolumn to which is fixed an antigen specifically recognized by theantibodies, in particular a viral antigen of interest.

The monoclonal antibodies can be obtained by the hybridomas techniquethe general principle of which is recalled hereafter.

In a first step, an animal, generally a mouse, (or cells in culturewithin the framework of in vitro immunizations) is immunized with thepeptide composition of the invention or with the vector of the inventionas “antigen of interest”, the B lymphocytes of which are then capable ofproducing antibodies against said antigen. These antibody-producinglymphocytes are then fused with “immortal” myelomatous cells (murine inthe example) in order to produce hybridomas. From the thus-obtainedheterogeneous mixture of cells, a selection is then made of cellscapable of producing a particular antibody and multiplying indefinitely.Each hybridoma is multiplied in clone form, each leading to theproduction of a monoclonal antibody the recognition properties of whichvis-à-vis the antigen of interest can be tested for example by ELISA, byimmunotransfer in one or two dimensions, by immunofluorescence, or usinga biocaptor. The monoclonal antibodies thus selected are subsequentlypurified in particular according to the affinity chromatographytechnique described above.

The peptide compositions, the expression vectors, the nucleotidesequences coding for said polyprotein NS3/NS4 and said polypeptide NS5b,as well as the antibodies of the invention are particularly effectivefor the inhibition, prevention and control of the infection of patientscarrying the HCV virus, so that their use for the preparation of amedicament constitutes another object of the invention.

The present invention also relates to a pharmaceutical composition, inparticular a vaccine, containing as active ingredient the peptidecomposition of the invention, or an expression vector of the invention,or an expression vector comprising a nucleotide sequence coding for thepolyprotein NS3/NS4 with an expression vector comprising a nucleotidesequence coding for the polypeptide NS5b, or the nucleotide sequencescoding for said polyprotein NS3/NS4 and said polypeptide NS5b, saidnucleotide sequences corresponding to the sequences contained in theexpression vectors of the invention, placed under the control ofelements necessary to an expression constitutive of and/or induciblefrom said peptides, or at least one of the antibodies of the invention.

By elements necessary to an expression constitutive of the peptides, ismeant a promoter that is ubiquitous or specific to the eukaryotic cells.

As elements necessary to an expression inducible from the peptides,there can be mentioned the elements of regulation of the operon of E.coli for tetracycline resistance (Gossen M. et al., Proc Natl Acad SciUSA, 89: 5547-5551 (1992).

According to a particular embodiment of the invention, thepharmaceutical composition also contains a pharmaceutically appropriatevehicle. Of course, a person skilled in the art will easily determinethe nature of the pharmaceutically appropriate vehicle and the quantityof polypeptides to be used as a function of the constituents of thepharmaceutical composition.

The quantity and nature of the pharmaceutically appropriate vehicle canbe easily determined by a person skilled in the art. They are chosenaccording to the desired pharmaceutical form and method ofadministration.

The pharmaceutical compositions of the invention are appropriate fororal, sublingual, sub-cutaneous, intramuscular, intravenous, topical,local, intratracheal, intranasal, transdermal, rectal, intraocular,intra-auricular administration, said active ingredient being able to beadministrated in a unitary dosage form of administration.

The unitary dosage forms of administration can be for example tablets,gelatin capsules, granules, powders, solutions or injectable oralsuspensions, transdermal patches, forms of sublingual, buccal,intratracheal, intraocular, intranasal, intra-auricular or by inhalationadministration, forms of topical, transdermal, sub-cutaneous,intramuscular or intravenous administration, forms of rectaladministration, or implants. For topical administration, creams, gels,ointments, lotions or collyriums can be envisaged.

These galenic forms are prepared according to the usual methods of thefields considered.

Said unitary dosage forms are dosed in order to allow dailyadministration of 0.001 to 10 mg of active ingredient per kg of bodyweight, according to the galenic form.

There may be particular cases where higher or weaker dosages areappropriate; the scope of the invention is not exceeded by such dosages.According to usual practice, the dosage appropriate to each patient isdetermined by the doctor according to the method of administration, theweight and the response of the patient.

According to another embodiment of the invention, the present inventionalso relates to a method of treatment of the pathologies associated withthe hepatitis C virus, which comprises the administration, to a patient,of an effective dose of a medicament of the invention.

The pharmaceutical compositions of the invention preferably contain asactive ingredient one of the vectors of the invention or an expressionvector comprising a nucleotide sequence coding for the polyproteinNS3/NS4 with an expression vector comprising a nucleotide sequencecoding for the polypeptide NS5b, so that they are useful in prophylacticand therapeutic vaccination.

Prophylactic and therapeutic vaccination can be implemented by injectionof a vaccine based on one or more expression vectors of the invention,to the extent that the expression vector or vectors finally code for thepolyprotein NS3/NS4 and for the polypeptide NS5b as active ingredient,said injection being or being not followed by boosters. It can also beimplemented by injecting two different types of expression vectors ofthe invention, firstly an adenovirus, then a poxvirus, simultaneously orat different times, and vice versa.

These vectors can be contained in a pharmaceutical kit.

Also, another object of the invention is pharmaceutical kits, inparticular vaccinal, comprising at least one expression vectorcomprising a nucleotide sequence coding for the polyprotein NS3/NS4 andat least one expression vector comprising a nucleotide sequence codingfor the polypeptide NS5b.

Another object of the invention is pharmaceutical kits, in particularvaccinal, comprising at least one expression vector of adenovirus typeas defined previously and/or at least one expression vector of poxvirustype as defined previously.

Prophylactic and therapeutic vaccination can also be implemented byinjection of a vaccine based on at least one expression vector of theinvention, or an expression vector comprising a nucleotide sequencecoding for the polyprotein NS3/NS4 with an expression vector comprisinga nucleotide sequence coding for the polypeptide NS5b, and at least onepharmaceutical composition of the invention constituted by the peptidecomposition of the invention or the antibodies of the invention. It canalso be implemented by injection of a vaccine based on at least oneexpression vector of the invention, or an expression vector comprising anucleotide sequence coding for the polyprotein NS3/NS4 with anexpression vector comprising a nucleotide sequence coding for thepolypeptide NS5b, and at least one nucleotide sequence coding for thepolyprotein NS3/NS4 and for the polypeptide NS5b.

Also, another object of the invention is pharmaceutical kits, inparticular vaccinal, comprising at least one expression vector of theinvention, or an expression vector comprising a nucleotide sequencecoding for the polyprotein NS3/NS4 with an expression vector comprisinga nucleotide sequence coding for the polypeptide NS5b, and at least onepharmaceutical composition of the invention or at least one nucleotidesequence coding for the polyprotein NS3/NS4 and for the polypeptideNS5b.

The present invention will be better understood using the followingexamples that are given only by way of illustration, and arenon-limitative, as well as using the attached FIGS. 1 to 7, in which:

FIG. 1A to 1K represents the maps of the different plasmids used forobtaining an adenovirus AdNS3NS4NS5b according to the invention, onwhich are indicated the sites of the different restriction enzymes andthe location of the sequence fragments coding for NS3/NS4 and for NS5b,

FIG. 2A to 2H represents the maps of the different plasmids used forobtaining a poxvirus MAV NS3NS4NS5b according to the invention, on whichare indicated the sites of the different restriction enzymes and thelocation of the sequence fragments coding for NS3/NS4 and pour NS5b,

FIG. 3 gives the cell response induced by the adenovirus AdNS3NS4,either according to the CTL test (FIG. 3A) where the epitope GLL wasused for stimulating the splenocytes in culture and for loading the CTLtargets and the result of which is expressed as a specific lysispercentage as a function of the effector/target ratio, or according tothe ELISPOT test (FIG. 3B), specific to the epitope GLL, where theresult is given in numbers of spots/10⁶ cells,

FIG. 4 gives the cell response induced by the adenovirus AdNS5baccording to the test ELISPOT, specific to the epitopes ALY and KLP,

FIG. 5 gives the cell response induced by the adenoviris AdCEIE2according to the CTL test where the epitope DLM was used for stimulatingthe splenocytes in culture and for loading the targets of the CTL andthe result of which is expressed as a specific lysis percentage as afunction of the effector/target ratio,

FIG. 6 gives the titre of the recombinant vaccinia virus, resulting fromthe trial test, in pfu/ml/mg ovary, for the 4 groups of 8 mice immunizedby the different combinations of adenovirus: AdNS3NS4+AdNS5b (1stgroup), the adenoviruses AdNS3NS4+AdNS5b+AdNS5a (2nd group), theadenoviruses AdNS3NS4+AdNS5b+AdCEIE2 (3rd group) and the adenovirusAdβGal (4th group) and

FIG. 7 gives the titre of the recombinant vaccinia virus, resulting fromthe trial test, in pfu/ml/mg ovary, for the 3 groups of 8 mice immunizedby the following different combinations of adenovirus: AdNS3NS4NS5b (1stgroup), AdNS3NS4+AdNS5b (2nd group) and AdβGal (3rd group).

EXAMPLE 1 Preparation of an Adenovirus Allowing the Expression of theProteins NS3/NS4 and NS5b According to the Invention

1. Adenovirus

The recombinant adenoviruses are generated by transfection (CaPO3) ofthe complementation line 293 (Graham, Smiley, et al. 1977) afterlinearization of the genomes by PacI. The recombinant viruses propagateand are amplified on this same line, and their purification is carriedout from the infected cells. The cells are recovered by centrifugation(1500 rpm, 10 minutes) and lysed by 3 freeze/thaw cycles. The celllysate is clarified by two centrifugations (2000 rpm, 10 minutes; 8000rpm, 15 minutes), then purified by two successive ultracentrifugations.The first is carried out on a Caesium Chloride gradient (densities 1.4and 1.25) at 30,000 rpm for 1 hour. The second is carried out on aCaesium Chloride cushion (density 1.34) at 35,000 rpm for 18 hours. Thephases containing the virions are removed and diluted by half in a 60%saccharose buffer. The viral suspensions are then dialysed againstformulation buffer (for 10 litres: 3423 g of saccharose; 12.11 g ofTris; 2.033 g of MgCl₂; 87.7 g of NaCl), then aliquoted. Their titrationis carried out by indirect immunofluorescence on 293 cells infected bydifferent viral dilutions and marked by an antibody specific to theadenoviral DNA-Binding Protein (α72K B6-8) (Reich, Sarnow, et al. 1983).

2. Preparation of the Adenovirus AdNS3NS4

This adenovirus allows the expression of the gene coding for thepolyprotein NS3/NS4 (SEQ ID No. 1 and 2) under the control of the CMVpromoter.

2.1 PCR Amplification of the Nucleotide Sequence Coding for thePolyprotein NS3/NS4

In order to do this, the following oligonucleotides were used:

oIV166: (SEQ ID No. 9) 5′-GGG GGG GCT ATG GCG CCT ATC ACG GCC TA-3′oIV171: (SEQ ID No. 10) 5′-GGG GGG ACG CGT TTA GCA TGG CGT GGA GCA GT-3′

as well as the following reagents:

Taq DNA Polymerase, PCR buffer, MgCl, 1.5 mM and dNTP 10 mM(Invitrogen).

The PCR conditions were the following:

5 minutes at 94° C., then

30 cycles of the series: 45 seconds at 94° C., 45 seconds at 62° C. and1 minute at 72° C., then

10 minutes at 72° C.

2.2 Insertion of the PCR Fragment NS3/NS4 into the Transfer PlasmidpTG13387

The following stages were carried out:

Enzymatic digestion of the plasmid pTG13387 (FIG. 1A, Transgene) byNheI/MluI (NheI, Invitrogen in React 4 Buffer and MluI, Invitrogen inReact 3 Buffer)

Enzymatic digestion of the fragment NS3/NS4 by NheI/MluI

Ligation (T4 DNA Ligase (Invitrogen) in Reaction Buffer (Invitrogen)),

Bacterial transformation (strain 5K, (Transgene)

Selection of bacterial clones on LB medium (Difco)+ampicillin (100μg/ml, Duchefa)

Plasmid maxi-preparation (Qiagen, according to manufacturer's protocol)of a positive clone after restriction analysis

Restriction analysis: digestion by SmaI (Invitrogen in React 4 Buffer)and obtaining of fragments of: 5450, 2164, 909, 214 and 180 pb

Obtaining of the plasmid pIV315 deleted from its E1 region andcontaining the sequence NS3/NS4 under the control of the CMV promoter(FIG. 1B).

2.3 Homologous Recombination with the Complete Adenoviral Genome Deletedfrom its E3 Region Contained in the Plasmid pTG6624

The following stages were carried out:

Enzymatic digestion of the plasmid pIV315 obtained above by PacI/PvuI(PacI in NEB1 buffer, Biolabs and PvuI in React 7 Buffer, Invitrogen);isolation on agarose gel of the fragment containing the cassettepCMV-NS3-NS4

Enzymatic digestion of the plasmid pTG6624 (FIG. 1C) by ClaI (in React 1Buffer, Invitrogen)

Bacterial transformation (strain BJ, (Transgene) in order to carry outthe homologous recombination between the two plasmid fragments

Selection of bacterial clones on LB medium+ampicillin (100 μg/ml)

Plasmid maxi-preparation (Qiagen) of a positive clone after restrictionanalysis

Restriction analysis: digestion by SmaI and obtaining of fragments of:2263, 621, 3814, 214, 2164, 909, 180, 2463, 6480, 1398, 4456, 1455,3540, 3386, 230 and 3685 pb

Obtaining of the complete adenoviral genome Adenovirus AdNS3NS4, deletedfrom its E3 and E1 regions, the latter having been replaced by theexpression cassette pCMV-NS3-NS4 (pIV317, FIG. 1D).

3. Preparation of the Adenovirus AdNS3NS4NS5b

This adenovirus allows the expression of the gene coding for thepolyprotein NS3/NS4 under the control of the CMV promoter and theexpression of the gene coding for the polypeptide NS5b under the controlof the SV40 promoter.

3.1 Construction of the Transfer Plasmid Allowing the Cloning in the E3Region of the Adenovirus of a Coding Sequence Under the Control of theCMV Promoter

The following stages were implemented:

Enzymatic digestion of the plasmid pTG4664 (FIG. 1E, Transgene) by BglII(in React 3 Buffer, Invitrogen)

Enzymatic digestion of the plasmid pTG3074 (FIG. 1F, Transgene) byBamHI/BglII (in React 3 Buffer, Invitrogen)

Ligation (T4 DNA ligase), bacterial transformation (strain 5K)

Selection of bacterial clones on LB medium+ampicillin (100 μg/ml)

Plasmid maxi-preparation (Qiagen) of a positive clone after restrictionanalysis

Restriction analysis: digestion by SmaI and obtaining of fragments of:4940, 1305 and 230 pb

Obtaining of the plasmid pIV267 (FIG. 1G)

Digestion of the plasmid pIV267 thus obtained by ClaI/MunI (in React 1Buffer, Invitrogen)

Treatment by DNA Polymerase I, Large (Klenow) Fragment (in React 2Buffer, Invitrogen)

Ligation (T4 DNA Ligase)

Bacterial transformation (strain 5K)

Selection of bacterial clones on LB medium+ampicillin (100 μg/ml)

Plasmid maxi-preparation (Qiagen)

Restriction analysis: digestion by SmaI and obtaining of fragments of:4692, 1305 and 230 pb

Obtaining of the plasmid pIV270, transfer plasmid allowing the cloningin the E3 region of the adenovirus of a coding sequence under thecontrol of the CMV promoter (FIG. 1H).

3.2 Replacement of the CMV Promoter by the SV40 Promoter in pIV270

The following stages were carried out:

PCR amplification of the nucleotide fragment corresponding to the SV40promoter, from the commercial plasmid pcDNAHygro (Clonetech) using thefollowing oligonucleotides:

oIV232: (SEQ ID No. 11) 5′-GGG GGG AGA TCT CCA GCA GGC AGA AGT ATG-3′oIV233: (SEQ ID No. 12) 5′-GGG GGG GTC GAC CGA AAA TGG ATA TAC AAGCTC-3′and according to the procedure described in point 2.1 above, except thata temperature of 58° C. instead of 62° C. was used

Enzymatic digestion of pIV270 by BglII/SalI (in React 10 Buffer,Invitrogen)

Enzymatic digestion of the PCR fragment by BglII/SalI

Ligation (T4 DNA ligase), bacterial transformation (strain 5K)

Selection of the bacterial clones on LB medium+ampicillin (100 μg/ml)

Plasmid maxi-preparation (Qiagen) of a positive clone after restrictionanalysis

Restriction analysis: digestion by SmaI and obtaining of fragments of:4692, 719, 80 and 230 pb

Obtaining of the plasmid pIV330, transfer plasmid allowing the cloningin the E3 region of the adenovirus of a coding sequence under thecontrol of the SV40 promoter (FIG. 11).

3.3 Insertion of the PCR Fragment NS5b into the Transfer Plasmid pIV330

The following stages were carried out:

PCR amplification of the nucleotide sequence coding for the protein NS5b(SEQ ID No. 3 and 4) using the following nucleotides:

oIV212: (SEQ ID No. 13) 5′-GGG GGG TCT AGA ATG TCA ATG TCC TAC ACA TGGAC-3′ oIV218: (SEQ ID No. 14) 5′-GGG GGG TCT AGA TTA CCG GTT GGG GAG CAGGT-3′and according to the procedure described in point 2.1 above, except thata temperature of 60° C. instead of 62° C. was used

Enzymatic digestion of the plasmid pIV330 obtained above by XbaI (inReact 2 Buffer, Invitrogen)

Enzymatic digestion of the PCR fragment by XbaI

Ligation (T4 DNA Ligase), bacterial transformation (strain 5K)

Selection of the bacterial clones on medium LB+ampicillin (100 μg/ml)

Plasmid maxi-preparation (Qiagen) of a positive clone after restrictionanalysis

Restriction analysis: digestion by SmaI and obtaining of fragments of:4692, 1505, 760, 719 and 230 pb

Obtaining of the plasmid pIV336, transfer plasmid in the E3 deletioncontaining the sequence NS5b under the control of the SV40 promoter(FIG. 1J)

3.4 Homologous Recombination with the Recombinant Adenoviral GenomepIV317 in Order to Obtain the Adenovirus of the Title

The following stages were implemented:

Digestion of the plasmid pIV317 obtained in point 2.3 above by SrfI (inUniversal Buffer, Stratagene)

Digestion of the plasmid pIV336 obtained in point 3.3 by NheI/SacII (inBuffer T, Amersham Pharmacia Biotech) and isolation on agarose gel ofthe fragment containing the cassette pSV40-NS5b

Bacterial transformation (strain BJ) for carrying out the homologousrecombination between the two plasmid fragments

Selection of the bacterial clones on medium LB+ampicillin (100 μg/ml)

Plasmid maxi-preparation (Qiagen) of a positive clone after restrictionanalysis

Restriction analysis: digestion by SmaI and obtaining of fragments of:6480, 4456, 3814, 3540, 3386, 2739, 2463, 2263, 2164, 1455, 1398, 1105,909, 760, 719, 621, 230, 214 and 180 pb

Obtaining of the desired complete adenoviral genome, deleted from the E1region, the latter having been replaced by the expression cassettepCMV-NS3-NS4, and deleted from the E3 region, the latter having beenreplaced by the expression cassette pSV40-NS5B (plasmid pIV342, FIG.1K).

4 Confirmation of the Expression of the Antigens Inserted into theDifferent Adenoviruses

The expression of the HCV antigens encoded by the adenoviruses AdNS3NS4,AdNS5b and AdNS3NS4NS5b was verified by Western blot after infection ofHuh7 cells.

As expected, all the antigens were expressed.

EXAMPLE 2 Preparation of a Poxvirus Allowing the Expression of theProteins NS3/NS4 and NS5b According to the Invention

1. MVA Poxvirus

The strain Modified Virus Ankara MVATG N33 was supplied by TRANSGENE S.A. (Strasbourg, France).

2. Preparation of the Transfer Plasmid Allowing the Expression of theGene NS3/NS4 Under the Control of the ph5r Promoter

2.1 Construction of the pIV250 Vector Containing the Recombination ArmsBRG2 and BRD2 of the MVA, as well as the selection gene GPT Under theControl of the Promoter ph5r (MVA), Followed by a Second Promoter ph5rin Order to Allow the Expression of the Gene of Interest

At this point, the insertion of the fragment ph5r-GPT-BRG3-ph5r(originating from the plasmid pTG9997, Transgene) into the plasmidpTG6018 (Transgene) containing the recombination arms BRG2 and BRD2 isdesired.

In order to do this, the following stages were carried out:

Enzymatic digestion by BamHI/SacI (in React 2 Buffer, Invitrogen) of thevector pTG6018 (FIG. 2A)

Enzymatic digestion by BamHI, then partial digestion by SacI of theplasmid pTG9997 (FIG. 2B)

Purification according to the QIAGEN protocol of the restrictionfragment of 1047 pb that contains the sequence coding forph5r-GPT-BRG3-ph5r

Ligation (T4 DNA Ligase), bacterial transformation (strain TG1,Statagene)

Selection of the bacterial clones on ampicillin (100 μg/ml)

Plasmid maxi-preparation (Qiagen) of a positive clone after restrictionanalysis (EcoRV+HindIII (in React 2 Buffer, Invitrogen): fragments of246, 439, 476, 826 and 2789 pb; SacI: fragments of 915 and 3861 pb)

Obtaining of the plasmid aimed at (pIV250, FIG. 2C).

2.2 PCR Amplification of the Nucleotide Sequence Coding for thePolyprotein NS3/NS4

The following oligonucleotides were used:

oIV225: (SEQ ID No. 15) 5′-GGG GGG CTG CAG ATG GCG CCT ATC ACG GCC TA-3′oIV226: (SEQ ID No. 16) 5′-GGG GGG TCT AGA TTA GCA TGG CGT GGA GCA GT-3′and according to the procedure described in Example 1, point 2.1 above,except that a temperature of 52° C. instead of 62° C. was used.

2.3 Insertion of the Fragment of PCR NS3-NS4 in the Plasmid pIV250

In order to do this, the following stages were carried out:

Enzymatic digestion of the plasmid pIV250 obtained in point 2.1 above byPstI (in React 2 Buffer, Invitrogen)/XbaI

Enzymatic digestion of the PCR fragment NS3/NS4 by PstI/XbaI

Ligation (T4 DNA Ligase), bacterial transformation (strain TG1)

Selection of the bacterial clones on ampicillin (100 μg/ml)

Plasmid maxi-preparation (Qiagen) of a positive clone after restrictionanalysis: (HindIII (in React 2 Buffer, Invitrogen): fragments of 4763and 2789 pb; SphI (in React 6 Buffer, Invitrogen): 1534 and 5991 pb;NcoI (in React 3 Buffer, Invitrogen): 2764 and 4761 pb)

Obtaining of the transfer plasmid containing the sequence coding for thepolyprotein NS3/NS4 under the control of the promoter ph5r (pIV327, FIG.2D).

3. Preparation of the Plasmid pIV328 Allowing the Expression of theProtein NS5b Under the Control of the p7.5 Promoter

3.1 PCR Amplification of the Nucleotide Sequence Coding for the ProteinNS5b

The following nucleotides were used:

oIV227: (SEQ ID No. 17) 5′-GGG GGG GTC GAC ATG TCA ATG TCC TAC ACA TGGAC-3′ oIV228: (SEQ ID No. 18) 5′-GGG GGG GCA TGC TTA CCG GTT GGG GAG CAGGT-3′and according to the procedure described in Example 1, point 2.1 above,except that a temperature of 52° C. instead of 62° C. was used.

3.2 Obtaining of the Plasmid

The following stages were carried out:

Enzymatic digestion of the PCR fragment coding for NS5b by SalI/SphI

Enzymatic digestion of pTG186 (FIG. 2E, Transgene) by SalI/SphI

Dephosphorylation of the vector pTG186 (ROCHE alkaline phosphatase)

Ligation (T4 DNA Ligase), bacterial transformation (strain TG1)

Selection of the bacterial clones on ampicillin (100 μg/ml)

Plasmid maxi-preparation (Qiagen) of a positive clone after restrictionanalysis: (HindIII: fragments of 1984, 2627 and 4437 pb; BglII:fragments of 321, 557, 1361, 1451, 2237 and 3121 pb; KpnI (in React 4Buffer, Invitrogen): fragments of: 2787 and 6261 pb)

Obtaining of the transfer plasmid containing the sequence coding for thepolypeptide NS5b under the control of the p7.5 promoter (pIV328, FIG.2F).

4. Preparation of the Transfer Plasmids pIV329 and pIV344 Allowing theExpression of the Gene Coding for the Polyprotein NS3/NS4 Under theControl of the ph5r Promoter and of the Gene Coding for the PolyproteinNS3/NS4 under the Control of the p7.5 Promoter

In order to do this the following stages were implemented:

PCR amplification of the nucleotide sequence coding for the protein NS5bfrom the plasmid pIV328 obtained in point 3.2 above using the followingoligonucleotides:

oIV229: (SEQ ID No. 19) 5′-GGG GGG TCT AGA CCG GTA GTT CGC ATA TACATA-3′ oIV218: (SEQ ID No. 14) 5′-GGG GGG TCT AGA TTA CCG GTT GGG GAGCAG GT-3′and according to the procedure described in Example 1, point 2.1 above,except that a temperature of 52° C. instead of 62° C. was used.

Enzymatic digestion of the fragment of PCR by XbaI

Enzymatic digestion of the plasmid pIV327 obtained in point 2.3 above byXbaI

Ligation (T4 DNA Ligase), bacterial transformation (strain TG1)

Selection of the bacterial clones on ampicillin (100 μg/ml)

Plasmid maxi-preparation (Qiagen) of 2 positive clones after restrictionanalysis: (PstI: pIV329: fragments of 3033 and 6466 pb, pIV344: 4641 and4858 pb; ApaI (in React 4 Buffer, Invitrogen): pIV329: 454, 960 and 8085pb, pIV344: 454, 1418 and 7627 pb; NcoI: pIV329: 4269, 469 and 4761 pb,pIV344: 3053, 1685 and 4761 pb; SmaI: pIV329: 214, 2164, 1444 and 5677pb, pIV344: 214, 2164, 928 and 6193 pb)

Obtaining either of the transfer plasmid allowing the expression of thepolyprotein NS3/NS4 under the control of the ph5r promoter and of theprotein NS5b under the control of the p7.5 promoter, the 2 expressioncassettes being oriented in the same direction (pIV329, FIG. 2G), or ofthe transfer plasmid allowing the expression of the polyprotein NS3/NS4under the control of the ph5r promoter and of the protein NS5b under thecontrol of the p7.5 promoter, the 2 expression cassettes being orientedin opposite directions (pIV344, FIG. 2H).

5. Confirmation of the Expression of the Antigens Inserted into theDifferent Poxviruses

It was verified by Western blot, after infection of Huh7 cells with thepoxviruses concerned, that the poxviruses pIV329 and pIV344, containingthe sequences coding for the polyprotein NS3/NS4 and the polypeptideNS5b, expressed said HCV antigens.

EXAMPLE 3 Demonstration of the Immunogenicity of the Combination ofNS3/NS4 and NS5b

1. Immunization of Mice

HLA-A2.1 transgenic mice were immunized, once, by intramuscularinjection of at least one adenovirus chosen from the followingadenoviruses:

AdNS3NS4 prepared in Example 1 above (point 2.3),

AdNS5 prepared in Example 1 above (point 3.3),

AdNS5a prepare according to the procedure of Example 1, point 2, exceptthat the following nucleotide primers were used in order to amplify thenucleotide sequence coding for the polypeptide NS5a (SEQ ID No. 5 and6):

oIV172: (SEQ ID No. 20) 5′-GGG GGG GGT ACC ATG TCC GGC TCG TGG CTAAGG-3′, oIV173: (SEQ ID No. 21) 5′-GGG GGG TCT AGA TTA GCA GCA GAC GATGTC GTC-3′,in the PCR the temperature of 62° C. was replaced by 56° C., theenzymatic digestion of pTG 13387 and of the fragment NS5a wereimplemented by KpnI/XbaI, restriction analysis by digestion by SmaI ofpTG13387 producing fragments of 180 and 7251 pb and of pTG6624 producingfragments of 2263, 621, 5615, 180, 2463, 6480, 1398, 4456, 1455, 3540,3386, 230 and 3685 pb.

AdCE1 E2 according to the procedure of Example 1, point 2, except thatthe following nucleotide primers were used in order to amplify thenucleotide sequence coding for the core-E1-E2 polyprotein (also calledCE1CE2) (SEQ ID No. 7 and 8):

oIV62: (SEQ ID No. 22) 5′-GGG GGG GCT AGC ATG AGC ACA AAT CCT AAACCT-3′, oIV68: (SEQ ID No. 23) 5′-GGG GGG TCT AGA TCA GGC CTC AGC CTGGGC TAT-3′,in the PCR the temperature of 62° C. was replaced by 56° C., theenzymatic digestion of pTG13387 and of the fragment CE1CE2 wereimplemented by NheI/XbaI, restriction analysis by digestion by SmaI ofpTG13387 producing fragments of 163, 435, 2270, 180 and 5254 pb and ofpTG6624 producing fragments of 2263, 621, 3618, 163, 435, 2270, 180,2463, 6480, 1398, 4456, 1455, 3540, 3386, 230 and 3685 pb,

AdNS3NS4NS5b prepared in Example 1 above (point 3) and

AdβGal (Transgene),

according to the following protocol:

10⁹ pfu of AdNS3NS4 or

10⁹ pfu of AdNS5b or

10⁹ pfu of AdCEIE2 or

10⁹ pfu of AdNS3NS4 and 10⁹ pfu of AdNS5b or

10⁹ pfu of AdNS3NS4, 10⁹ pfu of AdNS5b and 10⁹ pfu of AdNS5a

10⁹ pfu of AdNS3NS4, 109 pfu of AdNS5b and 10⁹ pfu of AdCEIE2

10⁹ pfu of AdNS3NS4 NS5b or

10⁹ pfu of Adβ-Gal as control.

Before immunization, the expression of the HCV and β-Gal antigens by thedifferent adenoviruses used for the immunization were verified byWestern blot.

2. CTL and ELISPOT Tests

Fifteen days after injection, the cell response was analyzed byisolating the spleen cells (splenocytes) of the mice and a CTL test andan ELISPOT test were carried out as follows:

For the CTL test, these splenocytes were cultured on 24-well plates inthe presence of:

5 μM of the epitope GLL (GLLGCIITSL, SEQ ID No. 24) in the case of thesplenocytes originating from mice having received AdNS3NS4, 5 μM of theepitope ALY (ALYDVVSTL, SEQ ID No. 25) or 5 μM of the epitope KLQ(KLQDCTMLV, SEQ ID No. 26) in the case of the splenocytes originatingfrom mice having received AdNS5b or 5 μM of the epitope DLM (DLMGYIPLV,SEQ ID No. 27) in the case of the splenocytes originating from micehaving received AdCE1E2, said epitopes being in synthetic peptide form(Eurogentex) and,

10 U of murine recombinant interleukin 2 (Brinster et al., Hepatology2001) per ml in alpha minimum essential medium (αMEM) for 5 days. On the5th day, the restimulation stage was carried out, which consists ofadding naive mice splenocytes to the splenocytes in culture in thepresence of said epitopes over 2 days. On the 7th day, the CTL test wascarried out, which consists of bringing into contact the spienocytesfrom the immunized mice after 7 days of culture (effector cells) and EL4S3-Rob HDD cells loaded with 10 μM of said epitopes and labelled withCr⁵¹ (target cells). The specific cytotoxic activity of the effectorcells was determined by measuring, after 4 hours of incubation with thetarget cells, Cr⁵¹ released following lysis of the target cells using aγ-Cobra II counting apparatus (Packard, Rungis, France) The maximumspontaneous release from wells containing either medium alone, or lysisbuffer (HCl IN) was determined. The specific percentage of cytotoxicitywas calculated by the formula:

(release in the test−spontaneous release)/(maximum release−spontaneousrelease)×100.

The epitope-specific lysis was determined by the difference between thepercentage of specific lysis obtained in the presence or in the absenceof said epitopes.

The ELISPOT test was carried out by culturing the splenocytes for 48hours in Multiscreen 96-well plates (Millipore) previously coated withanti-interferon gamma antibodies (IFNγ) (10 μg/ml final). Thesplenocytes were cultured in the presence of 10 μM of the appropriateepitopes, as indicated above, and of 10 U of murine recombinantinterleukin 2 per ml in αMEM. For the positive control, the splenocyteswere cultured in the presence of concanavalin A (5 μg/ml). For thenegative control, the splenocytes were cultured either in the presenceof a non-specific peptide belonging to the capsid protein of HCV, ofsequence DLMGYIPLV (also called irrelevant peptide), or in medium alonewithout epitope. The wells were washed three times, with 0.05% PBS-Tweenthen PBS respectively, an operation followed by incubation for 2 hourswith anti-IFNγ antibodies from biotinylated mice. After washing, thewells were incubated for 1 hour with a streptavidine-horseradishperoxidase conjugate and the enzymatic activity was developed bydegradation of the AEC (aminoethylcarbazole) substrate. The spotsobtained were counted using a Zeiss ELISpot reader (Zeiss microscope inconjunction with the KS-ELISpot software).

The results are indicated in FIGS. 3 to 5 in which M corresponds tomouse and Neg. mouse corresponds to the control mouse.

These results demonstrate that

AdNS3NS4 clearly induces a cell-mediated response specific of theexpressed antigens, as illustrated in FIGS. 3A and 3B by the detectionof T lymphocytes specific to the epitope GLL contained in NS3.

AdNS5b clearly induces a cell-mediated response specific of theexpressed antigens, as illustrated in FIG. 4 by the detection of Tlymphocytes specific to the epitope ALY and KLQ contained in NS5b.

AdCEIE2 clearly induces a cell-mediated response specific of theexpressed antigens, as illustrated in FIG. 5 by the detection of Tlymphocytes specific to the epitope DLM contained in the Core protein.

3. In Vivo Trial Test Using a Recombinant Vaccinia Virus

In order to evaluate whether the specific immune responses induced bythe different adenoviruses were capable of inducing protection against ainfectious disease trial (“in vivo protection”), we subjected thevaccinated mice to such a trial.

The mice not being directly infectable by HCV, in order to link theinduction of a specific immune response and resistance to an infection,we used a recombinant vaccinia virus (strain WR) coding for thenon-structural proteins of HCV (NS2 to NS5b) in order to carry out thistrial. This recombinant vaccinia virus, after intra-peritoneal injectionof 10⁷ pfu in the mouse, will be replicated in the animal. Thereplication of this virus induces an immune response both specific tothe vaccinia antigens and specific to the HCV antigens, as it alsoexpresses the NS proteins of HCV. This specific response to the HCVantigens will be all the more effective and vigorous as the mice willhave already received a vaccine expressing the HCV antigens. In otherwords, the more the effective vaccination (in the present case carriedout with the recombinant adenoviruses) has been (i.e. the immune systemof the mice have been effectively “primed” by the vaccine), the strongerwill be the anti-HCV response generated after trial by the recombinantvaccinia virus and, consequently, the more the mice are “protected”against this trial. In practice, the lower the residual vaccinia viruscount in the mice, the more effective the protection or theneutralization due to the vaccination has been.

The neutralization of the vaccinia virus reflects both the cell responseinduced by the HCV proteins and by the vaccinia proteins. Theneutralization is evaluated by titration of the residual vaccinia virusfrom the ovaries of the animals as follows: the ovaries are removed 4days post-trial, sonicated, freeze-thawed 3 times then aftercentrifugation, successive dilutions of supernatant are titratedaccording to the lysis plaque technique (Murata et al., PNAS, vol. 100,p. 6753-6758) on Hutk-cells. The viral titres are determined inpfu/ml/mg of ovary.

4. Demonstration of Superior Protection of a Vaccination Combining ThePolyprotein NS3/NS4 and the Polypeptide NS5b.

The recombinant virus titre of the vaccine was determined for 4 groupsof 8 mice immunized by the following combinations of adenoviruses:AdNS3NS4+AdNS5b (1st group), AdNS3NS4+AdNS5b+AdNS5a (2nd group),AdNS3NS4+AdNS5b+AdCEIE2 (3rd group) and AdβGal (4th group).

The results, given in FIG. 6, are treated statistically on the basis ofthe Wilcoxon Mann-Whitney non-parametric test (Méthodes Statistiques ál'usage des médecins et des biologistes, Collection Statistique enBiologie et en Médecine, Flammarion Medecine Sciences, (D. Schwarz),1977), which is based on a comparison of the averages, and allows thecomparison of the values of two independent samples x and y.

This test is implemented as follows: all of the values of the two groupsx and y to be compared are classified in increasing fashion. A rank isthen allocated to each value, and the sum of the ranks is calculated. Wxand Wy are then obtained. A reference value called (Wx)_(t) (theoreticalvalue in the null hypothesis where Wx is not different from Wy) is thencalculated and linked by the ratio: n (N+1)/2, with n=number of micetested in group_(x) and N=number of mice tested in groups x and y.

If W_(x) is less than (Wx)_(t) (low residual level of vaccinia virus inthe mice), then it can be concluded that the neutralization resultingfrom the vaccination is significantly effective.

If we take the example of the group AdNS3NS4S5b denoted x compared withthe group AdβGal denoted y, we obtain the following values:

-   -   Wx=1+2+4+6+8+11+13+14=59 (8 mice tested)    -   Wy=3+5+7+9+10+12+15+16=77 (8 mice tested)

Under the null hypothesis, Wx is not different from Wy, the expectedvalue is: (Wx)_(t)=(1/2)*8*17=68

Wx<(Wx)_(t), which signifies that the values obtained in the groupAdNS3NS4NS5b are smaller than those obtained in the group AdβGal andthat the neutralization resulting from the vaccination is significantlyeffective.

The statistical values for the other groups of mice are indicated inTable 1 below:

TABLE 1 Group/AdβGal Wx (Wx)_(t) AdNS3NS4 + NS5b 52 68 AdNS3NS4 + NS5b +68 68 NS5a AdNS3NS4 + NS5b + 74 68 CE1E2

The values in Table 1 above show that only a vaccination of the mice bya combination of the Adenoviruses NS3NS4 and adenovirus NS5b is capableof inducing a significant neutralization of the replication of thevaccinia virus used in the trial with respect to the group of controlmice vaccinated by AdβGal. The vaccinations carried out using thecombinations comprising (AdNS3NS4+AdNS5b+AdNS5a) or(AdNS3NS4+AdNS5b+AdCE1 E2), do not result in a significant differencecompared with the group of control mice immunized by AdβGal.

These results therefore make it possible to demonstrate, unexpectedly,the superior protection of a vaccination combining the polyproteinNS3NS4 and the polypeptide NS5b.

5. Confirmation of the Protection of a Vaccination Combining thePolyprotein NS3NS4 and the Polypeptide NS5b Expressed Jointly by theSame Vector

The recombinant vaccinia virus titre was determined for 3 groups of 8mice immunized by the following combinations of adenoviruses:AdNS3NS4AdNS5b (1st group), AdNS3NS4+AdNS5b (2nd group), and AdβGal (3rdgroup).

The results, given in FIG. 7, are treated statistically on the basis ofthe Wilcoxon Mann-Whitney non-parametric test as described in theprevious experiment.

The statistical values for groups 1 and 2 compared to the control groupAdβGal are indicated in Table 2 below:

TABLE 2 Group/AdβGal Wx (Wx)_(t) AdNS3NS4NS5b 49 68 AdNS3NS4 + NS5b 5368

The values in Table 2 above show that the vaccination of the mice by anadenovirus coding both for the three antigens NS3, NS4 et NS5b, like thecombination of the Adenovirus NS3NS4 and Adenovirus NS5b, is capable ofinducing a significant neutralization of the replication of the vacciniavirus used in the trial with respect to the group of control micevaccinated by the AdenoβGal. This result confirms the protection of avaccination combining the polyprotein NS3/NS4 and the polypeptide NS5bexpressed jointly by the same vector.

1-17. (canceled)
 18. Peptide composition characterized in that itcomprises a polyprotein NS3/NS4 of the hepatitis C virus, as well as apolypeptide NS5b of the hepatitis C virus.
 19. Peptide compositionaccording to claim 18, characterized in that NS3 and/or NS4 and/or NS5boriginate from viruses of different genotypes.
 20. Peptide compositionaccording to claim 18, characterized in that NS3, NS4 and NS5b originatefrom a virus of the same genotype, preferably genotype 1b. 21.Expression vector characterized in that it comprises a nucleotidesequence coding for the polyprotein NS3/NS4 and a nucleotide sequencecoding for the polypeptide NS5b, as well as the means necessary to theirexpression.
 22. Expression vector according to claim 21, characterizedin that the nucleotide sequences code for a polyprotein and apolypeptide originating from viruses of different genotypes. 23.Expression vector according to claim 21, characterized in that thenucleotide sequences code for a polyprotein and a polypeptideoriginating from a virus of the same genotype, preferably the genotype1b.
 24. Expression vector according to claim 21, characterized in thatthis vector is an adenovirus.
 25. Expression vector according to claim24, characterized in that the genome of the adenovirus is modified so asto replace the E1 region by the expression cassette CMV-NS3-NS4 and toreplace the E3 region by the expression cassette SV40-NS5b. 26.Expression vector according to claim 21, characterized in that thisvector is a poxvirus.
 27. Expression vector according to claim 26,characterized in that the genome of the poxvirus is modified so as toinsert the expression cassette ph5r-NS3-NS4 and to insert the expressioncassette p7.5-NS5b.
 28. Microorganism or host cell transformed by anexpression vector as defined in claim
 21. 29. A method for theinhibition, prevention or control of an infection caused by thehepatitis C virus in an animal, preferably human, comprising the use ofa peptide composition that comprises a polyprotein NS3/NS4 of thehepatitis C virus, as well as a polypeptide NS5b of the hepatitis Cvirus, or of an expression vector as defined in claim 21, or of anexpression vector comprising a nucleotide sequence coding for thepolyprotein NS3/NS4 with an expression vector comprising a nucleotidesequence coding for the polypeptide NS5b, or nucleotide sequences codingfor said polyprotein NS3/NS4 and said polypeptide NS5b, said nucleotidesequences corresponding to the sequences contained in the expressionvectors as defined in claim 21, placed under the control of elementsnecessary to an expression constitutive of and/or inducible from saidpeptides.
 30. Pharmaceutical composition, in particular vaccine,comprising as active ingredient the peptide composition that comprises apolyprotein NS3/NS4 of the hepatitis C virus, as well as a polypeptideNS5b of the hepatitis C virus, or an expression vector as defined inclaim 21, or an expression vector comprising a nucleotide sequencecoding for the polyprotein NS3/NS4 with an expression vector comprisinga nucleotide sequence coding for the polypeptide.
 31. Pharmaceuticalcomposition according to claim 30, characterized in that it alsocomprises a pharmaceutically appropriate vehicle.
 32. Pharmaceuticalkit, in particular vaccinal, characterized in that it comprises at leastone expression vector comprising a nucleotide sequence coding for thepolyprotein NS3/NS4 and at least one expression vector comprising anucleotide sequence coding for the Polypeptide NS5b.
 33. Pharmaceuticalkit, in particular vaccinal, characterized in that it comprises: atleast one expression vector characterized in that it comprises anucleotide sequence coding for the polyprotein NS3/NS4 and a nucleotidesequence coding for the polypeptide NS5b, as well as the means necessaryto their expression, wherein said vector is an adenovirus; and at leastone expression vector characterized in that it comprises a nucleotidesequence coding for the polyprotein NS3/NS4 and a nucleotide sequencecoding for the polypeptide NS5b, as well as the means necessary to theirexpression, wherein said vector is a poxvirus.
 34. Pharmaceutical kit,in particular vaccinal, comprising at least one expression vector asdefined in claim 21, or at least one expression vector comprising anucleotide sequence coding for the polyprotein NS3/NS4 with anexpression vector comprising a nucleotide sequence coding for thePolypeptide NS5b, and (i) at least one peptide composition thatcomprises a polyprotein NS3/NS4 of the hepatitis C virus, as well as apolypeptide NS5b of the hepatitis C virus or (ii) at least onenucleotide sequence coding for the polyprotein NS3/NS4 and for thepolypeptide NS5b.